An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

Abstract

Source: Ghassemi, S., et al. Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy. J. Vis. Exp. (2019)

This video demonstrates a technique to manufacture chimeric antigen receptor (CAR) T cells ex vivo. Human T cells are incubated with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, along with a medium supplemented with interleukin-2 (IL-2), leading to T cell activation and proliferation. Transgenic lentiviral vectors are introduced, allowing the transgenic RNA to integrate into the host genome and express the transgene. This results in the transduced T cells expressing chimeric antigen receptors (CARs) on their surface. The T cells are harvested at defined time points to assess CAR expression and cryopreservation.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. T Cell Activation, Transduction, and Expansion

1. Activate fresh or cryopreserved primary human T cells by mixing with anti-CD3/CD28 magnetic beads (e.g., dynabeads) at a ratio of 3 beads per T cell in 6-well cell culture dishes. Culture T cells in X-VIVO 15 medium supplemented with 5% normal human AB serum, 2 mM L-glutamine, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and interleukin 2 (IL2, 100 units/mL). Maintain T cells at a concentration of 10⁶ T cells/mL during ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti CD3/CD28 dynabeads	Thermo Fisher	40203D
APC Mouse Anti-Human CD8	BD Biosciences	555369	RRID:AB_398595
APC-H7 Mouse anti-Human CD8 Antibody	BD Biosciences	560179	RRID:AB_1645481
CountBright Absolute Counting Beads,	Invitrogen	C36950
HEPES	Gibco	15630-080
Human AB serum	Valley Biomedical	HP1022
Human IL-2 IS, premium grade	Miltenyi	130-097-744
L-glutamine	Gibco	28030-081
Multisizer Coulter Counter	Beckman Coulter
X-VIVO 15	Gibco	04-418Q